Pseudomonas aeruginosa is an opportunistic human pathogen that is responsible for nearly 10% of hospital-acquired infections and is the primary cause of morbidity and mortality in cystic fibrosis patients. P. aeruginosa infections have high attributable death rates and are extremely difficult to treat due to widespread antibiotic resistance. Our long-term goal is to understand how this ubiquitous environmental bacterium activates a coordinated virulence program upon introduction into the human host. This information will facilitate the development of therapeutic strategies aimed at inhibiting P. aeruginosa virulence mechanisms. P. aeruginosa possesses a wide variety of virulence factors. The capacity to express many of these factors and to cause disease in an animal model of acute infection depends on the ability of the bacteria to produce the second messenger adenosine 3', 5'-cyclic monophosphate (cAMP). We hypothesize that CyaB, a membrane-anchored adenylate cyclase, initiates a P. aeruginosa virulence program by producing cAMP in response to host cues. CyaB activity is positively regulated by components of the P. aeruginosa Type IV pilus chemotaxis system and negatively regulated by the alginate exopolysaccharide regulatory pathway. In addition, environmental cues including calcium concentration, osmolarity and CO2 substantially affect CyaB-dependent cAMP production. Once produced, cAMP activates a cAMP-dependent transcription factor (Vfr) that controls virulence gene expression. The goal of this proposal is to determine the relationship between structure and function of CyaB and the kinetics of CyaB-dependent cAMP production during infection. The Specific Aims are as follows: 1) Characterize the structure of the amino-terminal CyaB membrane-anchoring domain (termed MASE2 for Membrane-Associated Sensor-2) and determine its role in CyaB localization, signal detection and catalytic function. Preliminary results indicate that the MASE2 domain is required for enzyme activation. 2) Investigate the mechanism and biological significance of inorganic carbon (bicarbonate and CO2)-stimulated CyaB catalytic activity in vitro and in vivo. CyaB is a member of the inorganic carbon-activated family of adenylate cyclases and the CyaB catalytic domain is stimulated by HCO3- in vitro and by increased extracellular CO2 when expressed in P. aeruginosa. 3) Determine whether CyaB activity is modulated by host cues during infection. CyaB-lacking mutants are attenuated in vivo and CyaB is activated under conditions that mimic the bacterial-host interaction in vitro. We will construct reporter strains to determine if bacterial intracellular cAMP fluctuations occur during cell culture infection and whether cAMP levels change with time and disease progression in a mouse model of acute pneumonia. The bacterium Pseudomonas aeruginosa is a major cause nosocomial infection and is the primary cause of morbidity and mortality in individuals with cystic fibrosis. The prevalence of P. aeruginosa infection in immunocompromised individuals coupled with increasing antibiotic resistance among P. aeruginosa strains represents an urgent public health issue. The goal of this proposal is to define the mechanisms controlling the production of cyclic AMP, a central regulator of P. aeruginosa virulence. This proposal will result in the identification and characterization of novel therapeutic targets in P. aeruginosa as well as other pathogenic bacteria with cAMP-dependent virulence programs.